Plants on the ISS

Earth and its inhabitants have changed over time. One constant, though, has been gravity. Plants on Earth evolved in gravity, and gravity has long been known to play a key role in plant development. However, experiments conducted in the functional absence of gravity onboard the ISS have yielded results that challenge underlying assumptions regarding the role of gravity in root development.

The ISS National Lab provides a unique platform in which researchers can study fundamental plant development processes without the masking effect of gravity. An understanding of plant structure and behavior from spaceflight experiments translates directly to understanding how these processes work on the ground, said Anna-Lisa Paul, CASIS investigator at the University of Florida.

“Taking gravity out of the equation gives us insight into the inherent mechanisms of how plants work,” Paul said. “And the better you understand that, the better equipped you are to design experiments on Earth to build better crops and expand productivity—in addition to being able to take plants with us when we leave Earth’s orbit for extended missions or colonies on Mars.”

Understanding Gravity’s Role

On Earth, the plant hormone auxin is involved in orienting plant roots to grow in the direction of the pull of gravity. Auxin flows down the centermost cells of the root toward the root tip and then back up through the outer layer of root cells. This flow pattern is referred to as the “reverse fountain” model. Scientists had assumed that gravity played a role in establishing this flow.

Media Credit: Image courtesy of Anna-Lisa Paul and Robert Ferl, University of Florida.

Thus, in the functional absence of gravity onboard the ISS, one would expect a disruption in the flow and, instead, a diffuse distribution of auxin in the root tip. To test this hypothesis, Paul and co-investigator Robert Ferl, also a researcher at the University of Florida, recently conducted two experiments onboard the ISS—CARA and APEX03-2.

The results of these experiments demonstrated that the flow and distribution of auxin in the gravity-sensing portion of the root is actually not dependent on gravity. Instead, the pattern of auxin flow is a fundamental mechanism of root growth inherent in plants. These results were published in the Nature Partner Journal npj Microgravity in January 2016.

Taking Plant Research Into Space

The CARA experiment (Characterizing Arabidopsis Root Attractions) was funded by CASIS and flown to the ISS in 2014, and the APEX03-2 experiment (Advanced Plant EXperiments) was funded by NASA Space Biology and flown to the ISS in 2015. In both experiments, Paul and Ferl examined green fluorescent protein-reporter gene expression in the plant Arabidopsis thaliana (a model organism for plant biology research) to compare the distribution of auxin in the root tips of plants grown on the ISS versus ground controls.

For each experiment, the spaceflight plants were placed in flight hardware to enable their growth in space, and the ground control plants were placed in a controlled environment chamber at NASA’s Kennedy Space Center. For CARA, square petri plates with seedlings were attached to an interior wall of the ISS and received diffuse ambient light. For APEX03-2, the plates were placed inside Veggie, a climate-controlled locker on the ISS, with LED lights directly above the plates. Some of the plates in each experiment were wrapped with black cloth to block out all light.

“Repetition is the key to real science success,” Ferl said. “For APEX03-2, we used plants similar to ones used in CARA, but we also advanced the science by using plants of different ages and genotypes. This let us be sure that what we observed happens on more than a single trip to space.”

Paul and Ferl used two methods to examine the distribution of auxin in the root tips. While the plants were onboard the ISS, Paul and Ferl worked with technicians at NASA’s Glenn Research Center (GRC) in Ohio to use the Light Microscopy Module (LMM) on the ISS to image the live plants in real time. Once the plants returned from flight, Paul and Ferl examined the preserved plants in their laboratory using confocal microscopy.

To image the plants with the LMM on the space station, an ISS crewmember would take a survey photo of each plate and insert the plate into the LMM. Using the survey photo as a guide, Paul and Ferl worked with GRC technicians to navigate the LMM to look at regions of interest. “It was an interesting challenge to conduct that kind of experiment,” Paul said. “You want to sit at the microscope and move the controls with your own hands, but instead you have to send computer scripts to the microscope up on the space station.”

Challenging Assumptions

In analyzing the results from CARA and APEX03-2, Paul and Ferl found that all of the plants grown on the ISS—regardless of light source, growth habitat, age, or genotype—had the same pattern of auxin distribution in the root tip as plants on Earth. Although the functional absence of gravity and the different light sources did affect root growth, the flow of auxin in root tips remained the same.

These results show that although auxin is a key messenger in determining a plant’s response to gravity, gravity does not play a role in establishing the distribution of auxin in root tips, as scientists had assumed. Thus, the “reverse fountain” flow of auxin in root tips is neither reverse nor a fountain, because gravity is not involved.

Rather, the flow of auxin is an inherent developmental feature of root growth. Other messengers may also be involved in regulating a plant’s responses to gravity.

“When you can take your experiment to a place where you no longer have to worry about the influence of gravity, it allows you to see many things you would not have been able to see before,” Paul said. “We found that the native structures or signals in a plant—even when there are no directional cues—still enable the root to grow away from where it is planted, and you would not be able to see that on the background of a gravity environment.”

Interestingly, a fluorescent reporter gene used in CARA that targets another hormone, cytokinin, did exhibit different patterns of distribution in the root tips of plants grown on the ISS versus ground controls. Future experiments will further explore the effect of gravity on cytokinin signaling and related root growth.

Enabling Discovery

Access to the microgravity environment of the ISS National Lab gives researchers the opportunity to make discoveries that are not possible on Earth. CASIS and NASA work closely together with science investigators to enable access to the ISS National Lab and to make sure experiments such as CARA and APEX03-2 are successful, said Trent Smith, NASA Project Manager for Veggie, the plant-growth facility that housed APEX03-2.

“The point of the space station is that it’s really out there on the frontier,” Smith said. “Frontiers are rich in discovery, and being on the frontier gives us the ability to be surprised and to advance knowledge.”

Increased access to the ISS National Lab also allows researchers to run series of experiments, much like they do in their laboratories on the ground, Paul said. “This has opened the door for us to ask increasingly more complex and interesting questions that have an impact not only on growing plants in space, but also on our fundamental understanding of how plants respond to any novel environment.”

LMM

The LMM on the ISS features a modified Leica RXA microscope capable of using most standard Leica objectives. The LMM is configured to operate automatically and can also be controlled by the ISS crew or remotely from the ground. The LMM is capable of high-resolution black and white microscopy using bright field, epifluorescent, and fluorescent techniques.

Growing Toward Gravity

If you rotate a vertically growing plant by 90 degrees, the roots will turn back down and continue to grow down toward gravity. This is because when the plant is turned, a chain reaction causes the auxin to redistribute from the root tip and accumulate on the side of the root facing downward. This triggers growth on the opposite side of the root, making the root tip curve back down.

CONFOCAL MICROSCOPY

Confocal microscopy uses a laser to make high-resolution images of a specimen at multiple focal planes (or depth). The images are then assembled to reconstruct the 3-D structure of the specimen. Images can be taken as deep into the specimen as the light can penetrate. Future planned capabilities for the LMM on the ISS include confocal microscopy.